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Clinical Cancer Research Vol. 8, 1014-1018, May 2002
© 2002 American Association for Cancer Research

Clinical Trials

Immunization of Cancer Patients with a HER-2/neu, HLA-A2 Peptide, p369–377, Results in Short-lived Peptide-specific Immunity1

Keith L. Knutson2, Kathy Schiffman, Martin A. Cheever and Mary L. Disis

Division of Oncology, University of Washington, Seattle Washington, 98195-6527 [K. L. K., K. S., M. A. C., M. L. D.], and Corina Corporation, Seattle, Washington 98104 [M. A. C.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ideally, vaccines should be designed to elicit long-lived immunity. The goal of this study was to determine whether HER-2/neu peptide-specific CD8+ T-cell immunity could be elicited using an immunodominant HER-2/neu-derived HLA-A2 peptide alone in the absence of exogenous help. Granulocyte macrophage colony-stimulating factor (GM-CSF) was used as adjuvant. Six HLA-A2 patients with HER-2/neu-overexpressing cancers received 6 monthly vaccinations with a vaccine preparation consisting of 500 µg of HER-2/neu peptide, p369–377, admixed with 100 µg of GM-CSF. The patients had either stage III or IV breast or ovarian cancer. Immune responses to the p369–377 were examined using an IFN-{gamma} enzyme-linked immunosorbent spot assay. Before vaccination, the median precursor frequency (range), defined as precursors per 106 peripheral blood mononuclear cell, to p369–377 was 0 (no range). After vaccination, the median precursor frequency to p369–377 in four evaluable patients was 0 (0–116). Overall, HER-2/neu peptide-specific precursors developed to p369–377 in two of four evaluable subjects. The responses were short-lived and not detectable at 5 months after the final vaccination. Immunocompetence was evident, because patients had detectable enzyme-linked immunosorbent spot responses to tetanus toxoid and influenza. These results demonstrate that HER-2/neu MHC class I epitopes can induce HER-2/neu peptide-specific IFN-{gamma}-producing CD8+ T cells. However, the magnitude of the responses were low, as well as short-lived, suggesting that CD4+ T-cell help is required for lasting immunity to this epitope.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The CTL3 is a major mediator of antitumor immunity. Many tumor antigens have been discovered within the last decade, from which multiple MHC class I-restricted epitopes have been identified (1) . One such tumor antigen that is overexpressed on several cancers including breast and ovarian cancers is HER-2/neu, the gene product of erbB2/neu proto-oncogene (2, 3, 4) . Of several HLA-class I binding peptides identified within the HER-2/neu protein, the HLA-A2, 9-aa peptide, KIFGSLAFL (p369–377) has been more extensively characterized in both human preclinical and clinical studies. p369–377 was originally identified by Fisk et al. (5) as the immunodominant epitope recognized by four of four ovarian cancer-associated CTL lines and clones. In other studies, T cells reactive to p369–377 were derived from the tumor-associated lymphocytes of ovarian cancer (6) . In addition, splenocytes from HLA-A2 and human CD8 transgenic mice that were vaccinated previously with p369–377 recognized HLA-A2+, HER-2/neu+ human tumor lines (7) .

We have demonstrated previously that breast and ovarian cancer patients generate CD8 T-cell immunity to the p368–377 after vaccination with a helper peptide-based vaccine that consisted of HLA-class II peptides, each encompassing an HLA-A2 binding motif (8) . One of the vaccine peptides, p369–384, contained fully nested within its sequence the HLA-A2 binding peptide p369–377. Vaccination of patients with this helper peptide vaccine using GM-CSF as adjuvant resulted in the generation of an immunity to the p369–377 in 62% of patients. Importantly, the response to p369–377 was maintained in individuals followed for at least 1 year after the final immunization.

In the present study we evaluated whether active immunization with the HLA-A2 class I epitope p369–377, in the absence of helper peptide and using GM-CSF as adjuvant, would generate CD8 T-cell peptide responses in vivo and whether immunity generated would persist after active immunization has ended. The long-term goal of our studies is to develop a vaccine strategy that would generate long-term protective immunity against recurrence of tumor.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Clinical Trial.
Between April 1998 and July 1998, six patients with breast (n = 4) or ovarian cancer (n = 2) were enrolled in a Phase I HER-2/neu peptide-based vaccine trial approved by the University of Washington’s Human Subjects Division and the United States Food and Drug Administration. All of the subjects had received definitive conventional therapy for their disease. Eligibility was dependent on subjects: (a) having a WBC count >3.5 dl/ml; (b) having HER-2/neu protein overexpression in the primary tumor or metastasis; (c) being off of immunosuppressive drugs and chemotherapy for at least 30 days before enrolling; and (d) being HLA-A2 positive. All of the patients signed a protocol-specific consent. Patients received monthly vaccinations with 500 µg of an HLA-A2, 9-aa, HER-2/neu-derived peptide, p369–377, admixed with 125 µg/ml recombinant human GM-CFS (kindly supplied by Immunex Corporation, Seattle, WA) as an adjuvant. The vaccine preparation was divided into two intradermal injections administered monthly for 6 months to the same draining lymph node site. Subjects underwent peripheral blood draws or leukapheresis before and 30 days after each vaccination for immunological monitoring.

Materials.
The two peptides used in this study, either for immunization or in vitro use or both, were HLA-A2 influenza peptide GILGFVFTL (9) and p369–377 KIFGSLAFL (5) . The two peptides used for in vitro immunological monitoring were manufactured by either United Biochemical Inc. (Seattle, WA) or Multiple Peptide Systems (San Diego, CA), and all were >95% pure as assessed by high-performance liquid chromatography and mass-spectrometric analysis. The peptide used in vaccine preparations was manufactured by Multiple Peptide Systems (kindly provided by Corixa Corp., Seattle, WA) and approved for use in humans. Ficoll-Hypaque was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). RPMI 1640, HBSS, and PBS were purchased from Life Technologies, Inc. (Rockville, MD) and EHAA-120 from Biofluids (Rockville, MD). Human AB+ serum was obtained from Valley Biomedical, Incorporated (Winchester, VA), sterile nitrocellulose-backed microfiltration 96-well plates from Millipore Corp (Bedford, MA), and streptavidin-alkaline phosphatase and alkaline phosphatase colorimetric reagents were from Bio-Rad (Hercules, CA). Purified anti-IFN-{gamma} (clone # 1-D1K) and biotin-conjugated anti-IFN-{gamma} (clone # 7-B6-1) were purchased from Mabtech AB (Nacka, Sweden). HLA testing was performed by the Puget Sound Blood Bank (Seattle, WA). Tetanus toxoid was derived from Lederle Laboratories (Pearl River, NY).

Preparation of PBMCs.
PBMCs were obtained by either leukapheresis or 180–250-ml blood draws and isolated by density gradient centrifugation as described previously (10) . Cells were analyzed immediately or aliquoted and cryopreserved in liquid nitrogen in freezing medium (90% fetal bovine serum and 10% DMSO) at a cell density of 25–50 x 106 cells/ml.

ELIspot.
A 10-day ELIspot assay was used to determine precursor frequencies of peptide-specific CD8 T lymphocytes as described previously (8 , 11) . Resultant spots were then enumerated using a dissecting microscope. Precursor frequencies were calculated by subtracting the mean number of spots obtained from the no antigen control wells from the mean number obtained in the experimental wells. Statistical analysis was performed using the Student’s t test (Microsoft Excel 97). Precursor frequencies to peptide antigens were also enumerated from peripheral blood from HLA-A2+ healthy volunteers for comparison purposes. Assay validation was established in preliminary studies using the HLA-A2 influenza peptide over a PBMC range of 1.0–3.5 x 105 cells and also with the use of IFN-{gamma}-coated polystyrene beads (8 , 11) . These preliminary studies demonstrated that the assay is linear and precise between 2.0 and 3.5 x 105 PBMC/well, has a detection limit of 1:100,000, and has a detection efficiency of 93%. A positive response was defined as a precursor frequency that was both significantly (P < 0.05) greater than the mean of no antigen control wells and detectable (i.e., >1:100,000).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patients Immunized with a HER-2/neu 9-aa Peptide, p369–377, Increase Peptide-specific T-Cell Precursors.
Six subjects were enrolled on trial, the median age was 48 years (range, 46–73), and the median time from last chemotherapy was 5 months (range, 3–22). Of the breast cancer patients, three were diagnosed with stage V disease and one with stage IIIB disease. Of the ovarian cancer patients, one had stage III and the other had stage V disease. All four of the breast cancer patients received all six of the vaccinations. The two ovarian cancer patients withdrew early because of progressive disease. Toxicity was assessed using the National Cancer Institute Common Toxicity Criteria. There were no adverse events.

Patients enrolled in the study trial were assessed for immunocompetence using IFN-{gamma} ELIspot analysis evaluating the precursor frequencies to the HLA-A2 influenza peptide and whole tetanus toxoid (Fig. 1)Citation . These values were compared with those of 10 healthy HLA-A2+ volunteers. The mean influenza peptide-specific T-cell precursor frequency defined as peptide-specific precursors/106 PBMC of volunteer donors was 58 (range 0–333) and of patients was 44 (range 0–138). The mean values did not differ significantly from each other (P = 0.4). The mean tetanus toxoid precursor frequency of volunteer donors was 510 (range 0–1030) and that of patients was 359 (range 70–639). The mean values for tetanus toxoid did not differ significantly from each other (P = 0.2).



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  		Fig. 1. Patients are immunocompetent and demonstrate immunity to viral and bacterial antigens that is similar to healthy normal volunteers. Shown are the IFN-{gamma} ELIspot responses to influenza HLA-A2 peptide (circles) and tetanus toxoid (squares) in healthy HLA-A2 volunteers (open symbols) and cancer patients (closed symbols). Each symbol represents a measurement from a single unique subject calculated from six replicates. The solid lines indicate the mean precursor frequencies (precursors/106 PBMC) for the group.

 
Generation of T-cell precursors to the p369–377 was also evaluated patients using the IFN-{gamma}-based ELIspot assay, defined as p369–377 peptide-specific precursors/106 PBMC (Fig. 2)Citation . In addition, the p369–377-specific T-cell precursor frequency was also examined in the 10 normal HLA-A2 volunteer donors. Two of the 10 volunteer donors (20%) demonstrated immunity to p369–377, similar to studies published previously (5) . Before vaccination, p369–377 peptide-specific precursors were detectable in none of the six subjects. After immunization CD8 T-cell responses were detected to p369–377 in two of four evaluable subjects (mean 33, range 0–116).



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  		Fig. 2. Patients immunized with a HER-2/neu 9-aa peptide, p369–377, increase peptide-specific T-cell precursors. IFN-{gamma} ELIspot responses to p369–377 are shown for healthy normal HLA-A2 volunteers ({blacksquare}) and study subjects (•), with the mean delineated by a bar. Pre- and postimmunization ELIspot responses (precursors/106 PBMC). Each symbol represents a measurement from a single unique subject calculated on six replicates.

 
HER-2/neu HLA-A2 9-aa Peptide-specific Immunity Is Not Maintained after Active Immunizations Have Ended.
We analyzed the two patients who had detectable responses after the final vaccination over an extended period of time to determine whether CD8 peptide-specific T-cell responses were maintained or increased after active immunization (Fig. 3)Citation . Patient 2159 was a 52-year-old woman diagnosed in 1990 with lymph node-negative invasive ductal carcinoma of the left breast. The patient was disease-free after mastectomy and chemotherapy until 1997 when she presented with recurrent disease, which had metastasized to regional lymph nodes. The patient was treated to a minimal residual disease state with surgery, chemotherapy, and radiotherapy after which she began the immunizations in June 1998. This patient developed a detectable but low level (16 precursors/million) p369–377 response at the time of the 6th vaccine, which had diminished to undetectable levels 3 months later. In January 1999 the patient had relapsed.



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  		Fig. 3. HER-2/neu HLA-A2 9-aa peptide-specific immunity is not maintained after active immunizations have ended. Data shown are an IFN-{gamma} ELIspot time course (days) analysis of immunized patients, 9465 ({blacksquare}) and 2159 (•). Each symbol represents the mean of six replicates ± SE.

 
Patient 9465 was a 48-year-old woman who received a diagnosis of stage IIIB inflammatory carcinoma of the left breast in November 1996, which was treated into remission with both chemo- and radiotherapy. She started the p369–377 immunizations in April 1998. At the time of the 6th vaccine she demonstrated a p369–377-specific response of 116 precursors/million PBMCs. At 2 months after the last vaccination, the patient still maintained a response (93 precursors/million PBMCs) to the peptide, which was completely diminished by 5 months. The patient remained disease-free at time of last follow-up visit in September 2001.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Generation of a cytolytic T-cell response is thought to be essential for an effective antitumor response, and many studies of tumor immunity have focused primarily on the identification of HLA class I epitopes for tumor-associated antigens, including MAGE-1 (12) , NY-ESO-1 (13) , HER-2/neu (14) , tyrosinase (15) , and gp-100 (16) . The identification of HLA class I epitopes has resulted in several clinical trials to assess the feasibility and efficacy of cancer vaccination with individual antigen-specific HLA class I peptides. However, the major problem associated with immunization with a single HLA class I peptide is that it often results in only low-level, short-lived responses. Low-level, short-lived responses generated by HLA class I immunization could be because of either immune incompetence of the patient or the lack of help provided through activation of other components of the immune system, such as CD4 T cells. Ideally, vaccines should be designed to elicit high-level persistent immunity.

One potential reason why peptide-specific T-cell responses are low-level and short-lived after HLA class I peptide immunization is that tumor-bearing cancer patients can be immunosuppressed. Scheibenbogen et al. (17) have demonstrated that the presence of antigen-specific immune reactivity in melanoma patients is correlated with disease being in remission. Whereas many peptide vaccine studies are conducted in patients who have significant disease burden, in the present study the patients had either minimal or nondetectable disease and an excellent performance status. Therefore, the inability to generate immune responses was not likely because of patient immune incompetence. In fact, the data presented here demonstrate that patients had significant preexistent immunity to viral and bacterial antigens and were, thus, able to generate and maintain an immune response. The levels of viral precursors measured in the present study are consistent with those observed by Scheibenbogen et al. (17) in a cohort of melanoma and noncancer-bearing patients.

Tumor antigen-specific CD8 T cells are typically short-lived in vivo, as was demonstrated recently in a study by Dudley et al. (18) , who observed that GP-100-specific T-cell clones infused into melanoma patients rapidly disappeared within 2 weeks after infusion. Significant levels of CD8 peptide-specific immunity were also not maintained in the current study potentially because of the lack of a concomitant CD4 T-cell response. We hypothesize that the simultaneous generation of a CD4 T-cell helper response to the same tumor antigen during vaccination can enhance the longevity of the CD8 T-cell response. The requirement for CD4 T-cell help is supported by our previous findings that the long-lived CD8 T-cell immunity, including immunity to HER-2/neu p369–377, can be achieved by using a vaccine strategy that supplies both the CD8 T-cell epitope and the CD4 T-helper cell epitope simultaneously (8) . In a clinical trial performed recently patients were vaccinated with longer (15–18 amino acids) HER-2/neu helper peptides, each of which contained, fully nested within their sequences, an HLA-A2 binding motif. More than 75% of patients immunized with HER-2/neu helper peptides, using GM-CSF as an adjuvant as was done in this study, generated HER-2/neu peptide-specific CD8 T cells that persisted for at least 1 year after the final vaccination. Both the magnitude and the durability of the CD8 T-cell responses correlated with the magnitude of the CD4 T-helper cell responses generated by immunizing with longer peptides. However, the results of this current study should be interpreted with caution because of the small population size. Indeed, only with a randomized and controlled clinical trial could a conclusion be reached about whether a CD8 T-cell epitope vaccine containing a CD4 T-helper cell epitope is superior to a vaccine containing only a CD8 T-cell epitope. Murine antiviral and antitumor models have also defined the important role of CD4 T cells in maintaining a persistent CTL response (19 , 20) .

In addition to the generation of a tumor antigen-specific CD4 T-cell response, other strategies for providing T-cell help have shown promise. Dhodapkar et al. (21) reported increased durability of the immune response to an influenza matrix, HLA-A2-binding peptide in some subjects vaccinated with peptide-pulsed dendritic cells, suggesting that dendritic cells can initiate an environment capable of sustaining CD8 T cells. Conjugation of HLA class I peptides to foreign helper peptides or proteins has also been shown to enhance the immune response to HLA class I antigens. For example, Muderspach et al. (22) reported that 10 of 16 (63%) subjects demonstrated HPV-specific immunity after vaccination with an HPV-derived HLA-A2 peptide conjugated to the foreign helper peptide, PADRE-965.10. Similar results have also been observed by including the foreign protein keyhole limpet hemocyanin within the vaccine (23) . Although still in its early stages of development another promising method of boosting immunity to class I peptides, is to include CpG motifs as an adjuvant. Davila and Celis (24) showed recently that the CpG enhanced immune responses, 10–100-fold during immunization with MHC class I epitopes in murine models.

In summary, immunization of cancer patients with a HER-2/neu HLA-A2 peptide vaccine resulted in the generation of low-level peptide-specific CD8 T-cell immunity that did not persist for an extended time after active immunization had ended. Vaccination with HLA-class I peptides will likely require additional antigen-specific or nonspecific helper activity to generate long-lived immunity. Strategies that may improve outcome include using peptide-pulsed dendritic cells rather than soluble peptide, adding foreign helper epitopes, or by increasing adjuvant potency with CpG motifs.


   ACKNOWLEDGMENTS
 
We thank the patients who participated in this study and the oncologists who referred their patients to us. We also thank Lynne Fitzsimmons, for expert nursing care and protocol coordination.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a fellowship from the Department of Defense Breast Cancer Program (to K. L. K.) and by grants from the NIH, National Cancer Institute (R01 CA75163), and Department of Defense Breast Cancer Program (to M. L. D.). Patient care was conducted through the Clinical Research Center Facility at the University of Washington which is supported through NIH Grant MO1-RR-00037. Back

2 To whom requests for reprints should be addressed, at 1959 NE Pacific Street, HSB BB1361, Box 356527, Oncology, University of Washington, Seattle, WA 98195-6527. Phone (206) 221-5417; Fax: (206) 685-3128; E-mail: kknutson{at}u.washington.edu Back

3 The abbreviations used are: CTL, cytolytic CD8 T cell; GM-CSF, granulocyte macrophage colony-stimulating factor; PBMC, peripheral blood mononuclear cell; ELIspot; enzyme-linked immunosorbent spot; 9-aa, 9-amino acid; CpG, cytosine-phosphorothiolated guanine. Back

Received 12/ 7/01; revised 1/22/02; accepted 1/31/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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